PROTON NMR INVESTIGATION OF SUBSTRATE-BOUND HEME OXYGENASE - EVIDENCE FOR ELECTRONIC AND STERIC CONTRIBUTIONS TO STEREOSELECTIVE HEME CLEAVAGE
PROTON NMR INVESTIGATION OF SUBSTRATE-BOUND HEME OXYGENASE - EVIDENCE FOR ELECTRONIC AND STERIC CONTRIBUTIONS TO STEREOSELECTIVE HEME CLEAVAGE
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DOI：
10.1021/bi00187a033
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发表时间：
1994-05-31
期刊：
影响因子：
2.9
通讯作者：
LAMAR, GN
中科院分区：
文献类型：
--
作者：
HERNANDEZ, G;WILKS, A;LAMAR, GN
底物结合形式的酶血红素加氧酶（HO），催化立体特异性α-中桥分裂氯化血红素，产生胆绿素IX α，已被调查的H-1 NMR在其主要的高自旋和氰化物抑制低自旋形式。这两种衍生物产生的H-1 NMR光谱指示广泛的异质性，这在很大程度上是解决时，2倍对称的氯化血红素基板绑定。的异质性的结构起源被证明是由于类似于1：1异构体结合的天然氯化血红素基板的结合口袋。基板取向障碍是关于α，γ-介轴，如建立在2D NMR实验的基础上，确定特征性的芳香货车德瓦尔斯接触在基板结合口袋。异构体的底物HO复合物表现出不同的氰化物的亲和力，异构体的比例是敏感的氯化血红素2，4-取代基。通过同位素标记和2D NMR方法分配氯化血红素信号揭示了一种接触位移模式，其反映了一种不寻常的氯化血红素电子结构，其特征在于在给定的吡咯内的两个位置的离域自旋密度存在较大差异，而不是相邻吡咯之间更传统的较大差异。这种模式的自旋密度主要离域的吡咯位置相邻的α，γ-内消旋轴可以合理化，通过假定一个直接的电子扰动的血红素的蛋白质基质的阴离子侧链的形式接近α-内消旋碳。在铁卟啉化合物中已经观察到对氯化血红素电子结构的类似影响，其形式为化学取代中位，并且通过简单分子轨道理论成功地建模（Tan等人，1994年）。这被解释为HO的直接电子效应的证据，以激活的α-meso位置的亲电，而不是亲核攻击。独特的接触位移模式是目前不同程度的两个氯化血红素的方向，是强烈的pH值依赖性，并在很大程度上废除在酸性pH值。部分的几个血红素口袋残基的位置，它表明，这些残基的偶极位移的模式，这可能反映了远位空间的影响，协调氰化物的倾斜，显着不同的两个基板方向。可变的电子和空间的影响，两个氯化血红素底物方向上的蛋白质基质建议异质性，pH值依赖的动力学，但不变的立体选择性的酶反应，并可能占范围广泛的活性HO相对于各种周边修饰氯化血红素。
The substrate-bound form of the enzyme heme oxygenase (HO), which catalyzes the stereospecific alpha-meso bridge cleavage of hemin to yield biliverdin IX alpha, has been investigated by H-1 NMR in both its primarily high-spin and its cyanide-inhibited low-spin forms. Both derivatives yield H-1 NMR spectra indicative of extensive heterogeneity that is largely resolved when a 2-fold-symmetric hemin substrate is bound. The structural origin of the heterogeneity is shown to result from similar to 1:1 isomeric binding of the native hemin substrate in the binding pocket. The substrate orientational disorder is about the alpha,gamma-meso axis, as established on the basis of 2D NMR experiments that identify characteristic aromatic van der Waals contact in the substrate binding pocket. The isomeric substrate-HO complexes exhibit differential cyanide affinity, and the ratio of isomers is sensitive to the hemin 2,4-substituents. The assignment of hemin signals by isotopic labeling and 2D NMR methods reveals a contact shift pattern that reflects an unusual hemin electronic structure that is characterized by large differences in delocalized spin density for the two positions within a given pyrrole, rather than the more conventional large differences between adjacent pyrroles. This pattern of spin density delocalized primarily to the pyrrole positions adjacent to the alpha,gamma-meso axis can be rationalized by postulating a direct electronic perturbation of the hemin by the protein matrix in the form of an anionic side chain close to the alpha-meso carbon. Similar influences on hemin electronic structure, in the form of chemical substitution of the meso positions, have been observed in iron porphyrin compounds and successfully modeled by simple molecular orbital theory (Tan et al., 1994). This is interpreted as evidence for a direct electronic effect by HO to activate the alpha-meso position for electrophilic rather than nucleophilic attack. The unique contact shift pattern is present to different degrees for the two hemin orientations, is strongly pH dependent, and is largely abolished at acidic pH. Portions of several heme pocket residues are located, and it is shown that the pattern of the dipolar shifts for these residues, which likely reflects the distal steric influence on the tilt of the coordinated cyanide, differs significantly for the two substrate orientations. The variable electronic and steric influences of the protein matrix on the two hemin substrate orientations suggest heterogeneous, pH-dependent kinetics, but unaltered stereoselectivity in the enzyme reaction, and may account for the wide range of activity of HO with respect to a variety of peripherally modified hemins.